Optimization of the Isolation Protocol for Extracellular Vesicles (EVs) From Human Follicular Fluid

Summary

Background: Extracellular vesicles (EVs) are membrane-bound vesicles found in all biological fluids, containing various regulatory molecules, including microRNAs (miRNAs). It has been suggested that the EVs in human follicular fluid (the fluid surrounding the oocyte within the ovary) play a crucial role in oocyte development through these miRNAs. However, several methods exist for isolating these EVs from follicular fluid. Before further research can be conducted on the pathophysiology and potential diagnostic and therapeutic applications of these EVs and their content, the optimal isolation technique must be determined for future studies.

Each method has its advantages and disadvantages, and the purity and efficiency may vary between species and different biological fluids. There are only a limited number of studies comparing the various techniques for application in human follicular fluid.

Objective: This study will compare the three most commonly used techniques for isolating EVs: size-based, sedimentation-based (differential ultracentrifugation), and buoyancy-based isolation techniques.

Methodology:

In this prospective study, follicular fluid will be collected from four patients undergoing transvaginal oocyte retrieval following ovarian stimulation at Ghent University Hospital. Patients over 40 years of age or with endometriosis, adenomyosis, or PCOS (polycystic ovarian syndrome) will not be included in this study. During the oocyte retrieval procedure, follicular fluid will be collected from 2-4 oocytes per patient. Since follicular fluid is not used in the patient's treatment (residual material), it does not affect the treatment.

After collection, the three isolation techniques will be applied to each sample. The techniques will be compared in terms of purity (absence of contamination) and efficiency (EV concentration) using transmission electron microscopy, nanoparticle tracking analysis, and western blotting.

Conditions

• Extracellular Vesicles

Interventions

OTHER

Differential ultracentrifugation

Differential ultracentrifugation

OTHER

OptiPrep™ density gradient ultracentrifugation

OptiPrep™ density gradient ultracentrifugation

OTHER

Size-exclusion chromatography

Size-exclusion chromatography

OTHER

Combined techniques

Combined techniques

Sponsors & Collaborators

• University Ghent

  collaborator OTHER

• University Hospital, Ghent

  lead OTHER

Study Design

Allocation

NA

Purpose

BASIC_SCIENCE

Masking

NONE

Model

SINGLE_GROUP

Eligibility

Min Age

18 Years

Max Age

40 Years

Sex

FEMALE

Healthy Volunteers

No

Timeline & Regulatory

Start

2025-07-20

Primary Completion

2025-09-30

Completion

2025-12-31

Countries

• Belgium

Study Locations